Pharmaceutical composition of tricyclic compound

Abstract

The invention belongs to the technical field of medicines, and relates to a pharmaceutical composition of a tricyclic compound and a preparation method thereof, in particular to a pharmaceutical composition of a compound as shown in a formula (I) or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof. # imgabs0 #

Description

[0001] This application is a divisional application of the following application: Application date January 15, 2021; Application number 202180008484.2; Invention name: "Pharmaceutical composition of tricyclic PDE3 / PDE4 dual inhibitor compounds".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the rights and priority of Chinese invention patent application No. 202010042865.X filed with the State Intellectual Property Office of the People's Republic of China on January 15, 2020, the entire contents of which are hereby incorporated by reference into this document in their entirety. Technical Field

[0004] The present application belongs to the field of medical technology, and relates to a pharmaceutical composition of a tricyclic PDE3 / PDE4 dual inhibitor compound, a preparation method and uses thereof, and specifically to a pharmaceutical composition of a compound of formula (I) or a pharmaceutically acceptable salt thereof, a preparation method and uses thereof. Background Art

[0005] Phosphodiesterase (PDE) belongs to the superfamily enzyme system, which includes 11 families. Each family participates in different signal transduction and regulates different physiological processes. Among them, PDE3 is the main phosphodiesterase in human airway smooth muscle (ASM). Inhibition of PDE3 will increase the concentration of intracellular cAMP and relax bronchial smooth muscle. PDE4 plays a major regulatory role in the expression of pro-inflammatory and anti-inflammatory mediators. PDE4 inhibitors can inhibit the release of harmful mediators from inflammatory cells. Therefore, in principle, an inhibitor that inhibits both PDE3 and PDE4 will have both the bronchodilator effect (bronchodilation) of β-adrenergic receptor agonists and the anti-inflammatory effect (anti-inflammatory action) of inhaled glucocorticoids. The complementary dual-targeting functionality has a theoretical basis for greater efficacy than single targeting. It can achieve therapeutic effects that can only be achieved through combined medication through a single drug, overcome the defect that the physical and chemical properties of the drug components of combined medication cannot be fully matched, simplify the administration method, and facilitate quantitative administration.

[0006] Victoria Boswell et al, J.Pharmaco.Experi.Therap.2006,318,840-848 and WO200005830 reported compounds RPL554 and RPL565, which have long-acting bronchodilatory and anti-inflammatory effects, as well as physical and chemical properties such as poor solubility and high plasma clearance, and are suitable for inhalation administration. However, the data also show that their PDE4 inhibitory activity is not satisfactory and the anti-inflammatory effect is not ideal. Therefore, it is necessary to develop compounds that also have good PDE3 / 4 inhibitory activity.

[0007] Summary of the invention

[0008] In one aspect, the present application provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a surfactant:

[0009]

[0010] In some embodiments, the pharmaceutical composition further comprises a metal chelator (or metal complexing agent).

[0011] In some embodiments, the above pharmaceutical composition further comprises a buffer.

[0012] In some embodiments, the above pharmaceutical composition further comprises a diluent.

[0013] In some embodiments, the above pharmaceutical composition further comprises an osmotic pressure regulator.

[0014] In some embodiments, the above-mentioned pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, as well as a surfactant and a buffer.

[0015] In some embodiments, the above-mentioned pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, as well as a surfactant, a buffer and an osmotic pressure regulator.

[0016] In some embodiments, the above-mentioned pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, as well as a surfactant, a buffer and a metal chelator.

[0017] In some embodiments, the pharmaceutical composition described above comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, as well as a surfactant, a buffer, an osmotic pressure regulator and a metal chelator.

[0018] In some embodiments, the pharmaceutical composition described above comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, as well as a surfactant, a buffer, an osmotic pressure regulator, a metal chelator and a diluent.

[0019] In some embodiments, the pharmaceutical composition described above comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, and at least one of a buffer, an osmotic pressure regulator, a metal chelator and a diluent.

[0020] In some embodiments, the above-mentioned pharmaceutically acceptable salt is selected from maleate, sulfate, methanesulfonate or p-toluenesulfonate.

[0021] In some embodiments, the pharmaceutically acceptable salt of the compound of formula (I) above, wherein the molar ratio of the compound of formula (I) to the acid ion forming the pharmaceutically acceptable salt may be (1:1) to (1:2), for example 1:1 or 1:2.

[0022] In some embodiments, in the above-mentioned pharmaceutical composition, the "compound of formula (I) or a pharmaceutically acceptable salt thereof" can be replaced by "compound of formula (I)".

[0023] In some embodiments, the pharmaceutical composition comprises a compound of formula (I), a surfactant, a buffer, an osmotic pressure regulator, a metal chelator and a diluent.

[0024] In some embodiments, in the above-mentioned pharmaceutical composition, the concentration of the compound of formula (I) or its pharmaceutically acceptable salt is about 0.001 to about 80 mg / mL, preferably 0.002 to 50 mg / mL, and more preferably 0.1 to 20 mg / mL, calculated as the compound of formula (I).

[0025] The surfactant of the present application is a pharmaceutically acceptable surfactant, such as a wetting agent. The above-mentioned surfactant can be a nonionic surfactant, an anionic surfactant, a cationic surfactant or a zwitterionic surfactant. Preferably, the above-mentioned one or more surfactants are nonionic surfactants.

[0026] In some embodiments, the above-mentioned surfactant is one or more selected from ethylene glycol polyoxyethylene ether, polypropylene glycol alkyl ether, alkyl polyglucosides, octylphenol polyoxyethylene ether, alkylphenol polyoxyethylene ether, glycerol alkyl ester, polyoxyethylene sorbitan fatty acid ester (polysorbate), sorbitan alkyl ester, sorbitan fatty acid ester, cocamide MEA, cocamide DEA, dodecyl dimethyl amine oxide, block copolymers of polyethylene glycol and polypropylene glycol (poloxamers) and polyethoxylated tallow amine (POEA).

[0027] Preferably, the surfactant is one or more selected from polyoxyethylene sorbitan fatty acid esters (such as Tweens) and sorbitan fatty acid esters (such as Spans).

[0028] In some embodiments, the above-mentioned polyoxyethylene sorbitan fatty acid ester is one or more selected from polysorbate 20 (polyoxyethylene sorbitan laurate; Tween 20), polysorbate 40 (polyoxyethylene sorbitan monopalmitate), polysorbate 60 (polyoxyethylene sorbitan stearate) and polysorbate 80 (polyoxyethylene sorbitan monooleate; Tween 80).

[0029] In some embodiments, the sorbitan fatty acid ester is one or more selected from sorbitan monolaurate (Span 20), sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate and sorbitan monooleate.

[0030] More preferably, the above surfactant is selected from one or more of Tweens and Spans.

[0031] In some specific embodiments, the surfactant is selected from one or more of Tween 20, Tween 80 and Span 20.

[0032] In some embodiments, the concentration of the surfactant is about 0.01 to about 8 mg / mL. More typically, the concentration of the surfactant in the pharmaceutical composition is about 0.01 to 5 mg / mL, preferably about 0.02 to 3 mg / mL, more preferably about 0.05 to 2 mg / mL, and even more preferably about 0.1 to 1 mg / mL.

[0033] In some embodiments, in the above-mentioned pharmaceutical composition, the mass ratio between the compound of formula (I) or its pharmaceutically acceptable salt (calculated as the compound of formula (I)) and the surfactant is about 1:200 to 100:1, preferably about 1:150 to 50:1, more preferably about 1:50 to 25:1, further more preferably about 1:1 to 15:1, for example, it can be about 10:1.

[0034] In some embodiments, the above-mentioned buffer is a pharmaceutically acceptable buffer. The buffer can be any buffer suitable for a liquid pharmaceutical composition suitable for inhalation. The buffer is usually selected from one or more of sulfuric acid, hydrochloric acid, sodium hydroxide, citric acid, sodium citrate, lactic acid, sodium lactate, acetic acid, sodium acetate, trisodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, tartaric acid, sodium tartrate, glycine, boric acid, and phthalic acid. The preferred number of buffers is 2 and more, and the type is citrate buffer or phosphate buffer, more preferably the sodium salt of the two. Citrate buffer includes citric acid, sodium citrate and mixtures thereof. Phosphate buffer includes phosphoric acid, monosodium phosphate (i.e., sodium dihydrogen phosphate), disodium hydrogen phosphate and mixtures thereof.

[0035] In some embodiments, the buffer is selected from citric acid, citrate (eg, sodium citrate), tartaric acid, tartrate (eg, sodium tartrate), phosphoric acid, or phosphate (eg, sodium dihydrogen phosphate and disodium hydrogen phosphate).

[0036] In some embodiments, the buffer is selected from citrate (eg, sodium citrate), tartrate (eg, sodium tartrate), or phosphate (eg, sodium dihydrogen phosphate and disodium hydrogen phosphate).

[0037] In some embodiments, the concentration of the buffer is about 0.01 to about 50 mg / mL, preferably about 0.05 to about 40 mg / mL, more preferably 0.1 to about 25 mg / mL; and even more preferably 0.5 to about 6 mg / mL.

[0038] In some embodiments, the buffer is used to control the pH value of the pharmaceutical composition to be between about 3.0 and about 8.5, preferably between about 5 and about 7.

[0039] In some embodiments, the above-mentioned osmotic pressure regulator is generally selected from simple non-toxic salts, such as sodium chloride, potassium chloride, etc., or sugars, such as one or more of glucose, mannitol, xylitol, etc. In some embodiments, the above-mentioned osmotic pressure regulator is sodium chloride.

[0040] The concentration of the above-mentioned osmotic pressure regulating agent depends on the amount required to achieve isotonicity, such as isotonicity with plasma or lung fluid. The concentration of the osmotic pressure regulating agent is usually about 0.01 mg / mL to about 10 mg / mL, and more usually about 5 mg / mL to 9 mg / mL.

[0041] In some embodiments, the metal chelator is selected from one or more edetate salts such as edetic acid, edetate disodium, edetate calcium sodium, etc. Edetate salts (such as calcium salts, sodium salts) are preferred, and edetate disodium (EDTA-2Na) is particularly preferred.

[0042] The concentration of the metal chelator depends on the amount of metal ions that can be introduced during the preparation of the above-mentioned pharmaceutical composition, and is generally about 0.01 to about 40 mg / mL, preferably about 0.01 to about 20 mg / mL, more preferably about 0.01 to about 5 mg / mL; and more preferably about 0.01 to about 2 mg / mL.

[0043] In some embodiments, in the above-mentioned pharmaceutical composition, the diluent can be any pharmaceutically acceptable diluent. The diluent is suitable for inhalation administration. Usually, the diluent is selected from one or more of water, ethanol and glycerol. The preferred diluent is water, and the more preferred diluent is sterile water.

[0044] In some embodiments, in the above pharmaceutical composition, the amount of diluent used can be appropriate so that the concentration of the compound of formula (I) or its pharmaceutically acceptable salt or excipient in the above pharmaceutical composition is within a certain range.

[0045] In some embodiments, the pharmaceutical composition comprises a compound of formula (I) and a Tween or a Span; further, the Tween or Span is selected from one or more of Tween 20, Tween 80 and Span 20.

[0046] In some embodiments, the pharmaceutical composition further comprises phosphate; further, the phosphate can be selected from sodium dihydrogen phosphate or its monohydrate and disodium hydrogen phosphate; in some embodiments, the pharmaceutical composition further comprises sodium citrate or sodium tartrate.

[0047] In some embodiments, the pharmaceutical composition comprises a compound of formula (I), a mixture of a Span and a Tween (such as Tween 80, Tween 20 or Span 20) and water.

[0048] In some embodiments, the pharmaceutical composition comprises a compound of formula (I), a mixture of Spans and Tweens (such as Tween 80, Tween 20 or Span 20), sodium citrate or sodium tartrate and water.

[0049] In some embodiments, the pharmaceutical composition comprises a compound of formula (I), Tweens (such as Tween 80 or Tween 20), sodium dihydrogen phosphate or its monohydrate, disodium hydrogen phosphate, disodium edetate and water.

[0050] In some embodiments, the pharmaceutical composition comprises a compound of formula (I), Tweens (such as Tween 80 or Tween 20), sodium dihydrogen phosphate or its monohydrate, disodium hydrogen phosphate, sodium chloride, disodium edetate and water.

[0051] In some embodiments, the above pharmaceutical composition comprises a compound of formula (I), Tweens (such as Tween 80 or Tween 20), sodium citrate or sodium tartrate, sodium chloride and water.

[0052] In some embodiments, the pharmaceutical composition comprises a compound of formula (I), a mixture of Spans and Tweens (such as Tween 80, Tween 20 or Span 20), sodium citrate or sodium tartrate, sodium chloride and water.

[0053] In some embodiments, the above-mentioned pharmaceutical composition comprises a compound of formula (I), a surfactant, a buffer, an osmotic pressure regulator, a metal chelator and a diluent, wherein the concentration of the compound of formula (I) is 0.002-50 mg / mL, the concentration of the surfactant is 0.02 to 3 mg / mL, the concentration of the buffer is 0.1 to about 25 mg / mL, the concentration of the osmotic pressure regulator is 5 mg / mL to 9 mg / mL, and the concentration of the metal chelator is 0.01 to about 5 mg / mL.

[0054] In some embodiments, in the above pharmaceutical composition, the surfactant, buffer, osmotic pressure regulator, metal chelator and diluent are respectively as defined above.

[0055] In some embodiments, the above-mentioned pharmaceutical composition comprises a compound of formula (I), a surfactant, a buffer, an osmotic pressure regulator, a metal chelator and a diluent, wherein the concentration of the compound of formula (I) is 0.002-50 mg / mL, the concentration of the surfactant is 0.02 to 3 mg / mL, the concentration of the buffer is 0.1 to about 25 mg / mL, the concentration of the osmotic pressure regulator is 5 mg / mL to 9 mg / mL, and the concentration of the metal chelator is 0.01 to about 5 mg / mL; the surfactant is one or more of Tween 20, Tween 80 and Span 20, the buffer is one or more of sodium dihydrogen phosphate or its monohydrate, disodium hydrogen phosphate, tartaric acid and citric acid, the osmotic pressure regulator is sodium chloride, the metal chelator is one or more of edetate disodium and edetate sodium calcium, and the diluent is water.

[0056] In some embodiments, the above-mentioned pharmaceutical composition comprises a compound of formula (I), Tween 80, sodium dihydrogen phosphate or its monohydrate, sodium hydrogen phosphate, sodium chloride, disodium edetate and water; wherein the concentration of the compound of formula (I) is 0.002-50 mg / mL, the concentration of Tween 80 is 0.02 to 3 mg / mL, the concentration of sodium dihydrogen phosphate and disodium hydrogen phosphate is 0.1 to 25 mg / mL, the concentration of sodium chloride is 5 mg / mL to 9 mg / mL, and the concentration of disodium edetate is 0.01 to about 5 mg / mL.

[0057] In some embodiments, the pharmaceutical composition of the compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is in solid form; in some embodiments, the compound of formula (I) is a crystal of the compound of formula (I).

[0058] In some embodiments, the pharmaceutical composition of the compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is a product obtained by controlling the particle size of the crystals of the compound of formula (I).

[0059] In some embodiments, the crystal of the compound of formula (I) has an X-ray powder diffraction pattern using Cu Kα radiation with diffraction peaks at the following 2θ angles: 5.81±0.2°, 13.96±0.2°, 15.01±0.2°, 17.95±0.2° and 24.73±0.2°.

[0060] In some embodiments of the present application, the crystals of the compound of the above formula (I) have an X-ray powder diffraction pattern using Cu Kα radiation having diffraction peaks at the following 2θ angles: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 17.95±0.2°, 24.73±0.2° and 26.13±0.2°.

[0061] In some embodiments, the crystals of the compound of formula (I) above have an X-ray powder diffraction pattern using Cu Kα radiation having diffraction peaks at the following 2θ angles: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 20.83±0.2°, 24.73±0.2° and 26.13±0.2°.

[0062] In some embodiments, the crystal of the compound of formula (I) above has a diffraction peak at the following 2θ angles in its X-ray powder diffraction pattern using Cu Kα radiation: 5.81±0.2°, 8.38±0.2°, 9.13±0.2°, 11.16±0.2°, 11.60±0.2°, 12.82±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 18.91±0.2°, 20.83±0.2°, 24.36±0.2°, 24.73±0.2°, 25.78±0.2° and 26.13±0.2°.

[0063] In some embodiments, the crystals of the compound of formula (I) above comprise 5, 6, 7, 8, 9, 10, 11, 12 or more diffraction peaks selected from the following 2θ angles in its X-ray powder diffraction pattern using Cu Kα radiation: 5.81±0.2°, 8.38±0.2°, 9.13±0.2°, 11.16±0.2°, 11.60±0.2°, 12.82±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 18.91±0.2°, 20.83±0.2°, 24.36±0.2°, 24.73±0.2°, 25.78±0.2° and 26.13±0.2°.

[0064] In some embodiments, the crystals of the compound of formula (I) above have an X-ray powder diffraction pattern using Cu Kα radiation, comprising 5, 6, 7, 8, 9, 10 or 11 diffraction peaks selected from the following 2θ angles: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 20.83±0.2°, 24.73±0.2° and 26.13±0.2°.

[0065] In some embodiments, the crystals of the compound of formula (I) above have an X-ray powder diffraction pattern using Cu Kα radiation, comprising 5, 6, 7, 8 or 9 diffraction peaks selected from the following 2θ angles: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 17.95±0.2°, 24.73±0.2° and 26.13±0.2°.

[0066] In some embodiments, the crystals of the compound of formula (I) above have an XRPD spectrum using Cu Kα radiation, and the peak positions and relative intensities of the diffraction peaks are shown in Table 1 below:

[0067] Table 1

[0068] serial number 2θ angle (±0.2°) Relative strength (%) serial number 2θ angle (±0.2°) Relative strength (%) 1 5.81 47.9 17 17.69 20.9 2 8.38 23.7 18 17.95 69.7 3 9.13 15.4 19 18.91 15.0 4 10.57 8.2 20 20.21 8.0 5 11.16 46.9 21 20.83 24.3 6 11.60 17.6 22 21.25 5.3 7 11.77 6.2 23 22.96 9.9 8 12.82 16.5 24 24.14 5.1 9 13.96 99.5 25 24.36 19.5 10 14.47 38.4 26 24.73 100.0 11 15.01 80.7 27 25.48 11.1 12 15.71 8.0 28 25.78 19.9 13 16.03 9.1 29 26.13 61.8 14 16.54 9.2 30 29.05 12.5 15 16.76 24.4 31 29.37 6.3 16 17.47 8.3

[0069] In some embodiments, the crystal of the compound of formula (I) has an XRPD pattern using Cu Kα radiation such as Figure 1 shown.

[0070] In some embodiments, the crystal of the compound of formula (I) has an exothermic peak at 247.70°C±2°C in its differential scanning calorimetry curve.

[0071] In some embodiments, the crystal of the compound of formula (I) has a differential scanning calorimetry spectrum as shown in Figure 2 shown.

[0072] In some embodiments, the thermogravimetric analysis curve of the crystal of the compound of formula (I) above shows a weight loss of 0.4870% at 155.75±2°C; and a weight loss of 7.287% between 155.75±2°C and 262.18±2°C.

[0073] In some embodiments, the crystals of the compound of formula (I) above have a thermogravimetric analysis spectrum such as Figure 3 shown.

[0074] In some embodiments, in the above pharmaceutical composition, the particle size of the compound of formula (I) or its pharmaceutically acceptable salt is X 50 ≤10μm, preferably between 0.1μm and 8μm.

[0075] In some embodiments, in the above pharmaceutical composition, the particle size of the compound of formula (I) or its pharmaceutically acceptable salt is X 50 ≤5μm and X 90 ≤10μm.

[0076] The above-mentioned pharmaceutical compound of the present application can be administered to humans in various preparation forms, such as solutions, etc., which are intended to be formed into a suitable form for oral administration or inhalation.

[0077] In some embodiments, the pharmaceutical compositions of the present application are administered by inhalation.

[0078] In some embodiments, the pharmaceutical composition of the present application is administered by oral or nasal inhalation.

[0079] In some embodiments, the pharmaceutical composition of the present application is a solution for inhalation.

[0080] In some embodiments, the pharmaceutical composition of the present application is in the form of a suspension.

[0081] In some embodiments, the pharmaceutical composition of the present application is in the form of a suspension for inhalation.

[0082] On the other hand, the present application provides a method for preparing the above-mentioned pharmaceutical composition, comprising the following process: mixing a surfactant with a compound of formula (I) or a pharmaceutically acceptable salt thereof. Preferably, the preparation method comprises mixing a surfactant, a compound of formula (I) or a pharmaceutically acceptable salt thereof, and at least one selected from the following: a metal chelator, a buffer, a diluent, and an osmotic pressure regulator. More preferably, the present application provides a method for preparing the above-mentioned pharmaceutical composition, comprising the following process: mixing a wetting agent, a buffer, an osmotic pressure regulator, a metal chelator, a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a diluent.

[0083] In some embodiments, the preparation method of the above pharmaceutical composition comprises the following process:

[0084] 1) a wetting agent, a buffer, an osmotic pressure regulator, a metal chelating agent and a diluent are mixed to form a solution,

[0085] 2) The compound of formula (I) or a pharmaceutically acceptable salt thereof is mixed with the solution of step 1).

[0086] In some embodiments, the above preparation process further comprises step 3): homogenizing the product obtained in step 2).

[0087] In some embodiments, after the above homogenization process, the particle size of the compound of formula (I) or its pharmaceutically acceptable salt is X 50 ≤5μm and X 90 ≤10μm.

[0088] In some embodiments, the above preparation process further includes a filling step.

[0089] On the other hand, the present application also provides a method for preventing or treating a disorder associated with PDE3 and / or PDE4 in a mammal, comprising administering a therapeutically effective amount of the above pharmaceutical composition to a mammal, preferably a human, in need of such prevention or treatment.

[0090] On the other hand, the present application also provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing or treating a disease associated with PDE3 and / or PDE4.

[0091] On the other hand, the present application also provides use of the above pharmaceutical composition for preventing or treating disorders associated with PDE3 and / or PDE4.

[0092] On the other hand, the present application also provides the above pharmaceutical composition for preventing or treating disorders associated with PDE3 and / or PDE4.

[0093] In some embodiments of the present application, the disorder associated with PDE3 and / or PDE4 is selected from asthma or chronic obstructive pulmonary disease (COPD).

[0094] Technical Effects

[0095] The compound of formula (I) of the present application and its pharmaceutical composition have significant dual PDE3 and PDE4 inhibitory effects, and have significant inhibitory effects on TNF-α in human peripheral blood mononuclear cells (hPBMC), and also show excellent anti-inflammatory effects in LPS (lipopolysaccharide)-induced rat acute lung injury model; have high in vivo plasma clearance rate, low oral plasma systemic exposure and low oral bioavailability, and good safety of local administration; have low inhibitory effects on five isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomal cytochrome P450, and have no risk of drug-drug interaction; reduce the total number of BALF white blood cells, have significant anti-inflammatory effects, and have a low effective dose; and reduce the airway resistance index Penh.

[0096] The crystals of the compound of formula (I) and the crystals of the pharmaceutically acceptable salts thereof have advantages in terms of drug activity, pharmacokinetics, bioavailability, hygroscopicity, melting point, stability, solubility, purity, and ease of preparation, so as to meet the requirements of drug production, storage, transportation, and formulation, etc. The crystal form of the compound of formula (I) is not very hygroscopic, which is conducive to the absorption of inhaled drugs.

[0097] The pharmaceutical composition of the compound of formula (I) of the present application has good stability, no growth of impurities, and exhibits pharmaceutically acceptable levels of impurities; good dispersibility, kinetic stability, including no significant sedimentation of particles, no growth of particle size, and within the range required for effective delivery of approved inhalation products. In addition, the composition has uniform and moderate particle size and fast absorption rate. The delivery rate is good, the dosage is accurate, the proportion of aerosol particles that can be inhaled is high, and the dosage of fine particles is high.

[0098] Explanation of terms

[0099] Unless otherwise required in this application, throughout the specification and the claims that follow, the word "comprise" or "comprises" and its English variations such as "comprises" and "comprising" or equivalents should be interpreted as an open, inclusive sense, that is, "including but not limited to", meaning that in addition to the listed elements, components and steps, other unspecified elements, components and steps may also be included.

[0100] References to "one embodiment" or "an embodiment" or "in another embodiment" or "in certain embodiments" throughout this specification are intended to include in at least one embodiment the specific referenced elements, structures or features described in connection with that embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, the specific elements, structures or features may be combined in any appropriate manner in one or more embodiments.

[0101] It should be understood that the singular article "a", "an" and "the" used in the specification and the appended claims include plural objects unless the context clearly states otherwise; in other words, in this document, singular terms cover plural terms and vice versa. Thus, for example, a reaction mentioned including "a catalyst" includes one catalyst, or two or more catalysts. It should also be understood that the term "or" is generally used in its sense including "and / or" unless the context clearly states otherwise.

[0102] The term "treatment" means administering the compound or formulation described herein to improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

[0103] (i) inhibiting a disease or disease state, i.e. arresting its development;

[0104] (ii) ameliorating a disease or condition, even if causing regression of the disease or condition.

[0105] The term "prevention" means administering the compounds or formulations described herein to prevent a disease or one or more symptoms associated with the disease, including preventing a disease or disease state from occurring in a mammal, particularly when such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state.

[0106] The term "therapeutically effective amount" means an amount of the compound of the present application that (i) treats or prevents a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The amount of the compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and the present disclosure.

[0107] Typically, the particle size is quantified by measuring a characteristic equivalent spherical diameter (called volume diameter) by laser diffraction, for example as determined by a laser particle sizer.

[0108] The present application uses volume diameter (VD) to represent particle size distribution.

[0109] The term "X 10 " refers to the particle size corresponding to the volume cumulative distribution percentage reaching 10%. Its physical meaning is that particles smaller than it account for 10% of the total volume.

[0110] The term "X 50 " refers to the particle size corresponding to the volume cumulative distribution percentage reaching 50%, which is called volume median diameter. Its physical meaning is that particles smaller than it account for 50% of the total volume.

[0111] The term "X 90 " refers to the particle size corresponding to when the volume cumulative distribution percentage reaches 90%. Its physical meaning is that particles smaller than it account for 90% of the total volume.

[0112] Herein, unless otherwise indicated, each parameter value (including 2θ values, reaction conditions) is deemed to be modified by the term "about" to reflect the measurement error that exists for each value, for example, there is an error of ±5% relative to a given value.

[0113] For the purpose of description and disclosure, all patents, patent applications and other identified publications are expressly incorporated herein by reference. These publications are provided only because they are disclosed prior to the filing date of the present application. All statements regarding the dates of these documents or the representations of the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any reference to these publications herein does not constitute an admission that the publications are part of the common general knowledge in the art in any country. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 is the XRPD spectrum of the crystal of the compound of formula (I);

[0115] Figure 2 is the DSC spectrum of the crystal of the compound of formula (I);

[0116] Figure 3 is a TGA spectrum of the crystal of the compound of formula (I);

[0117] Figure 4 is a DVS diagram of the crystal of the compound of formula (I);

[0118] Figure 5 is the total number of leukocytes in BALF;

[0119] Figure 6 It is a lung function test (airway resistance index Penh) under methacholine MCh induction. DETAILED DESCRIPTION

[0120] The purpose of the following specific embodiments is to enable those skilled in the art to more clearly understand and implement the present application. They should not be considered as limiting the scope of the present application, but are merely exemplary descriptions and typical representatives of the present application.

[0121] Example

[0122] Synthesis of intermediate BB-1

[0123]

[0124] Step 1: Synthesis of compound BB-1-2

[0125] Under nitrogen atmosphere, a mixture of compound BB-1-1 (21.10 g) and ethyl cyanoacetate (11.00 g, 10.38 mL) was stirred at 100 ° C for 16 hours. After the reaction was completed, the mixture was cooled to 70 ° C, and ethanol (30 mL) was slowly added dropwise, and a large amount of solid precipitated. Filter, and dry the filter cake under reduced pressure to obtain the product BB-1-2.

[0126] 1 H NMR (400MHz, DMSO-d6) δ = 8.26 (t, J = 5.2Hz, 1H), 6.86 (d, J = 8.0Hz, 1H), 6.79 (br s,1H),6.71(d,8.0Hz,1H),4.00(q,J=6.8Hz,2H),3.72(s,3H),3.59(s,2H ),3.31-3.23(m,2H),2.64(t,J=7.2Hz,2H),1.32(t,J=6.8Hz,3H).MS-ESI m / z:263.1[M+H] + .

[0127] Step 2: Synthesis of compound BB-1-3

[0128] Under nitrogen atmosphere, phosphorus oxychloride (379.50 g, 230.00 mL) was heated to 85 ° C, and compound BB-1-2 (26.00 g) was added in batches. The reaction mixture was stirred at 85 ° C for 2 hours. After the reaction was completed, most of the phosphorus oxychloride was removed by distillation under reduced pressure. Dichloromethane (200 mL) was added to the residue and washed with water (100 mL × 2). After the organic phase was dried over anhydrous sodium sulfate, the desiccant was removed by filtration and concentrated under reduced pressure. The crude product was purified by slurrying with ethyl acetate (20 mL) to obtain compound BB-1-3.

[0129] 1 H NMR (400MHz, CD3OD) δ = 7.16 (s, 1H), 6.83 (s, 1H), 4.62 (s, 1H), 4.12 (q, J = 6.8Hz, 2H), 3 .86(s,3H),3.35(d,J=6.4Hz,2H),2.84(t,J=6.4Hz,2H),1.44(t,J=6.8Hz,3H).MS-ESI m / z:245.1[M+H] + .

[0130] Step 3: Synthesis of compound BB-1-4

[0131] At 0°C, compound BB-1-3 (1.00 g) was added in batches to 98% concentrated sulfuric acid (12.88 g, 128.69 mmol, 7.00 mL). The reaction mixture was stirred at 27°C for 3 hours. After the reaction was completed, the mixture was added to cold water (15 mL), and then sodium hydroxide aqueous solution (4 mol / L, 32 mL) was added dropwise to adjust the pH value to neutral, and then extracted with ethyl acetate (100 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to obtain compound BB-1-4.

[0132] MS-ESI m / z:263.1[M+H] + .

[0133] Step 4: Synthesis of compound BB-1-5

[0134] At 0°C, sodium (2.42 g) was added to ethanol (80 mL) in batches. After the mixture was stirred at 28°C for 0.5 hours, compound BB-1-4 (6.90 g) was added to the solution in batches and stirred at 80°C for 0.5 hours. Diethyl carbonate (9.32 g, 9.51 mL) was then added all at once, and the mixture was stirred at 80°C for 5 hours. After the reaction was completed, the mixture was cooled to room temperature, ice water (30 mL) was slowly added, and then the pH value was adjusted to neutral with dilute hydrochloric acid (2 mol / L, 53 mL). A large amount of solid precipitated and was filtered. The filter cake was purified by slurrying with ethanol (10 mL) to obtain compound BB-1-5.

[0135] 1 H NMR (400MHz, DMSO-d6) δ = 11.22 (br s, 1H), 7.35 (s, 1H), 6.95 (s, 1H), 6.22 (s, 1H), 4.09 (q, J = 6.8Hz, 2H), 3.90 (br s, 2H), 3.83 (s, 3H), 2.89 (br s,2H),1.35(t,J=6.8Hz,3H).MS-ESI m / z:289.1[M+H] + .

[0136] Step 5: Synthesis of compound BB-1-6

[0137] Compound BB-1-5 (5.00 g) was dissolved in phosphorus oxychloride (30 mL) at room temperature. The reaction mixture was stirred at 100 ° C for 16 hours under a nitrogen atmosphere. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure. Water (100 mL) was added and extracted with dichloromethane (150 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to obtain compound BB-1-6. MS-ESI m / z: 306.9 [M + H] + .

[0138] Step 6: Synthesis of compound BB-1

[0139] Compound BB-1-6 (925.67 mg) was dissolved in isopropanol (8 mL) at room temperature, and 2,4,6-trimethylaniline (2.10 g) was added. The reaction mixture was stirred at 90 ° C for 15 hours under a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by slurrying with ethanol (6 mL) to obtain compound BB-1.

[0140] 1H NMR(400MHz,DMSO-d6)δ=8.85(br s,1H),7.27(s,1H),6.97(s,1H),6.90(s,2H),6.45(s,1H),4.10(q,J=6.8Hz,2H),3.90(t,J=6.0H z,2H),3.86(s,3H),2.87(t,J=6.0Hz,2H),2.45(s,3H),2.11(s,6H),1.37(t,J=6.8Hz,3H).MS-ESI m / z:406.2[M+H] + .

[0141] Synthesis of compound BB-4

[0142]

[0143] Step 1: Synthesis of compound BB-4-1

[0144] Compound BB-1 (1.00 g) was dissolved in 2-butanone (35 mL) at room temperature, and 2-(2-bromoethyl)isoindoline-1,3-dione (3.76 g), potassium carbonate (3.07 g) and sodium iodide (2.22 g) were added in sequence. The reaction mixture was stirred at 85 ° C for 72 hours under a nitrogen atmosphere. After the reaction was completed, the mixture was concentrated to remove most of the organic solvent, and water (30 mL) and ethyl acetate (25 mL × 3) were added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure. The residue was purified by rapid silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 15:1 to 3:1) to obtain compound BB-4-1.

[0145] MS-ESI m / z:579.3[M+H] + .

[0146] Step 2: Synthesis of compound BB-4

[0147] Compound BB-4-1 (500.00 mg) was dissolved in chloroform (3 mL) and ethanol (3 mL) at room temperature, and hydrazine hydrate (152.67 mg, 85% purity) was added. The mixture was stirred at 28 ° C for 16 hours under a nitrogen atmosphere. After the reaction was completed, the mixture was concentrated to remove most of the organic solvent, and water (15 mL) and dichloromethane (15 mL × 3) were added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to obtain compound BB-4.

[0148] 1H NMR (400MHz, DMSO-d6) δ = 6.95 (s, 1H), 6.85 (br s,2H),6.66(s,1H),5.31(s,1H),4.14(t,J=6.8Hz,2H),4.05(q,J=6.8Hz,2H) ,3.91(t,J=6.4Hz,2H),3.62(s,3H),2.90-2.86(m,4H),2.22(s,3H),1.95(br s,6H),1.33(t,J=6.8Hz,3H).MS-ESI m / z:449.2[M+H] + .

[0149] Example 1: Preparation of the compound of formula (I)

[0150] 5-Hydroxy-3-methyl-1,2,3-triazole-4-carboxylic acid (18.50 mg) was dissolved in DCM (1 mL) at 20°C, HATU (8.80 mg) and triethylamine (57.40 μL) were added thereto, and the mixture was stirred for 2 hours, followed by the addition of compound BB-4 (50 mg), and the mixture was stirred for 16 hours at the same temperature. The mixture was diluted to 10 mL with DCM, washed with water (30 mL*3), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was separated and purified by prep-HPLC to obtain the target compound of formula (I) as a yellow solid.

[0151] 1 H NMR (400MHz, CD3OD) δ = 6.94 (s, 2H), 6.87 (s, 1H), 6.77 (s, 1H), 5.52 (s, 1H), 4.48 (t, J = 6.0Hz, 2H), 4.15 (s, 3H), 4.12-4.08 (m, 2H), 4 .01(t,J=6.0Hz,2H),3.87(t,J=6.0Hz,2H),3.69(s,3H),2.94(t,J=6.0Hz,2H),2.29(s,3H),2.06(s,6H),1.41(t,J=6.8Hz,3H).MS m / z[M+H] + 574.1.

[0152] Example 2: Preparation of crystals of the compound of formula (I)

[0153] 50 mg of the compound of formula (I) was added to a 4 mL glass bottle, 1 mL of anhydrous ethanol and 0.2 mL of water were added, and the temperature was raised to 40°C and stirred for 48 hours. The mixture was naturally cooled to room temperature, centrifuged and separated into solids, and vacuum dried to obtain 46 mg of solid crystals. The XRPD spectrum is shown in Figure 1 As shown in the DSC spectrum Figure 2As shown in the TGA spectrum Figure 3 shown.

[0154] Example 3-36

[0155] Preparation process:

[0156] 1) Add each auxiliary material into the preparation tank, stir and dissolve to obtain an auxiliary material solution;

[0157] 2) adding the compound of formula (I) to the excipient solution prepared above, and stirring to form a uniform suspension;

[0158] 3) further through a high pressure homogenizer, microfluidizer or sand mill to control the particle size of the compound of formula (I) in the preparation to X 50 ≤5μm and X 90 ≤10μm;

[0159] 4) Get the product.

[0160] The specific amounts of auxiliary materials and products are described in Table 2 below.

[0161] Table 2

[0162]

[0163] * In Examples 35 and 36, after the auxiliary materials in the above step 1) are mixed and dissolved, it is necessary to add an appropriate amount of sodium hydroxide to adjust the pH to 5.0 to 7.0.

[0164] Experimental Example 1: Study on the stability of the crystalline solid of the compound of formula (I)

[0165] High performance liquid chromatography (HPLC)

[0166] The chromatographic conditions of the HPLC method are shown in the table below:

[0167] Chromatographic column: Zorbax SB C-18, 4.6 mm × 150 mm, 5 μm (PDS-HPLC-007)

[0168] Mobile phase A: 0.1% TFA in water

[0169] Mobile phase B: 100% ACN

[0170] Sample preparation: The sample was dissolved in a mixed solvent of acetonitrile and water (acetonitrile: water = 50:50 (v / v)).

[0171] Solid Stability Lofting Method

[0172] The stability of the compound placed under the following conditions was investigated, and samples were taken at different time points for content detection. About 5 mg of the crystals of the compound of formula (I) prepared in Example 2 were accurately weighed and placed in a dry and clean glass bottle, in duplicate, spread into a thin layer, as the formal test sample, and placed under the test conditions of influencing factors ((60°C), (relative humidity 92.5%), light (total illumination 1.2×10 6 Lux·hr / Near UV 200w·hr / m 2 ), (40℃, relative humidity 75%), or (60℃, relative humidity 75%), the samples were completely exposed, covered with aluminum foil and pierced with small holes. Sampling and analysis were carried out at 5 days, 10 days, 1 month, 2 months, and 3 months. The samples placed under light (visible light 1200000Lux, ultraviolet 200W) conditions were completely exposed at room temperature. The experimental results are shown in Table 3.

[0173] Table 3 Solid stability sample content analysis results (5 days, 10 days, 1 month, 2 months, 3 months)

[0174] RRT / Norm% Total impurities% Day 0 0.11 60℃_5 days 0.10 60℃_10 days 0.11 92.5%RH_5 days 0.11 92.5%RH_10 days 0.10 Protect from light 0.11 illumination 0.10 40℃+75%RH-10 days 0.10 60℃+75%RH-10 days 0.10 40℃+75%RH-1 month 0.10 60℃+75%RH-1 month 0.10 40℃+75%RH-2 months 0.08 40℃+75%RH-3 months 0.09

[0175] It can be seen that the crystals of the compound of formula (I) of the present application have good stability under high temperature, high humidity or light conditions, and there is no growth of impurities during the test period.

[0176] Experimental Example 2: Study on the Hygroscopicity of Crystals of the Compound of Formula (I)

[0177] Instrument model: SMSDVS Advantage Dynamic Vapor Sorption Instrument

[0178] Test conditions: Take a sample (10-20 mg, the crystals prepared in Example 3) and place it in a DVS sample tray for testing.

[0179] The detailed DVS parameters are as follows:

[0180] Temperature: 25℃

[0181] Balance: dm / dt = 0.01% / min (minimum: 10min, maximum: 180min)

[0182] Drying: Drying at 0% RH for 120 min

[0183] RH(%) test level: 10%

[0184] RH (%) test step range: 0%-90%-0%. The obtained dynamic vapor adsorption (DVS) graph is as follows: Figure 4 shown.

[0185] Depend on Figure 4It can be seen that the crystals of the compound of formula (I) of the present application have low hygroscopicity.

[0186] Experimental Example 3: In vitro detection of the inhibitory activity of compounds on PDE 3A enzyme

[0187] Purpose: To measure AMP / GMP expression based on fluorescence polarization, i.e. tracer AMP / GMP antibody binding to indicate enzyme activity.

[0188] Reagents:

[0189] Assay buffer solution: 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.01% Brij 35, 1 mM dithiothreitol (DTT), and 1% DMSO.

[0190] Enzyme: Recombinant human PDE3A (gene accession number NM_000921; amino acid 669) was expressed by baculovirus in Sf9 insect cells with an N-terminal GST tag, and its molecular weight = 84 kDa.

[0191] Substrate: 1 μM cAMP

[0192] Detection: AMP2 / GMP2 antibody and AMP2 / GMP2 AlexaFluor633 tracer.

[0193] Steps:

[0194] 1. Dissolve the recombinant human PDE3A enzyme and substrate (1 μM cAMP) in freshly prepared experimental buffer;

[0195] 2. Transfer the above PDE3A enzyme buffer solution into the reaction well;

[0196] 3. Add 100% DMSO-dissolved compounds to the PDE3A enzyme buffer solution reaction wells by acoustic technology (echo 550; nanoliter range) and incubate at room temperature for 10 minutes;

[0197] 4. Add the substrate buffer solution to the above reaction wells to start the reaction;

[0198] 5. Incubate at room temperature for 1 hour;

[0199] 6. Add the assay mixture ( The reaction was terminated with AMP2 / GMP2 antibody and AMP2 / GMP2 AlexaFluor 633 tracer) and incubated for 90 minutes with slow mixing. The fluorescence polarization measurement range was Ex / Em=620 / 688.

[0200] Data analysis: The fluorescence polarization signal was converted into nM by calculating the percentage of enzyme activity relative to the DMSO control according to the AMP / GMP standard curve and Excel software. Curve fitting was performed using GraphPad Prism (drawing medical icons). The experimental results are shown in Table 4.

[0201] Experimental Example 4: In vitro detection of the inhibitory activity of compounds on PDE 4B enzyme

[0202] Purpose: To measure AMP / GMP expression based on fluorescence polarization, i.e. tracer AMP / GMP antibody binding to indicate enzyme activity.

[0203] Reagents:

[0204] Assay buffer solution: 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.01% Brij 35, 1 mM DTT, and 1% DMSO.

[0205] Enzyme: Recombinant human PDE4B (gene accession number NM_002600; amino acid 305) was expressed by baculovirus in Sf9 insect cells with an N-terminal GST tag, and its molecular weight = 78 kDa.

[0206] Substrate: 1 μM cAMP

[0207] Detection: AMP2 / GMP2 antibody and AMP2 / GMP2 AlexaFluor633 tracer.

[0208] Steps:

[0209] 1. Dissolve the recombinant human PDE4B enzyme and substrate (1 μM cAMP) in freshly prepared experimental buffer;

[0210] 2. Transfer the above PDE4B enzyme buffer solution into the reaction well;

[0211] 3. Add 100% DMSO-dissolved compounds to the PDE4B enzyme buffer solution reaction wells by acoustic technology (Echo 550; nanoliter range) and incubate at room temperature for 10 minutes.

[0212] 4. Add the substrate buffer solution to the above reaction wells to start the reaction

[0213] 5. Incubate at room temperature for 1 hour

[0214] 6. Add the assay mixture ( The reaction was terminated with AMP2 / GMP2 antibody and AMP2 / GMP2 AlexaFluor 633 tracer) and incubated for 90 minutes with slow mixing. The fluorescence polarization measurement range was Ex / Em=620 / 688.

[0215] Data analysis: Fluorescence polarization signals were converted to nM according to the AMP / GMP standard curve and the % enzyme activity relative to the DMSO control was calculated by Excel software. Curve fitting was performed using GraphPad Prism (Drawing Medical Icons).

[0216] The experimental results are shown in Table 4:

[0217] Table 4 Results of in vitro screening tests of compounds

[0218]

[0219] The active ingredients in the pharmaceutical composition of the present application have significant dual PDE3 and PDE4 inhibitory effects.

[0220] Experimental Example 5: Pharmacokinetic Study in Beagle Dogs

[0221] Male beagles were selected as test animals in this study. The LC-MS / MS method was used to quantitatively determine the drug concentration in the plasma of beagles at different time points after intravenous injection or oral administration of the compound of formula (I) to evaluate the pharmacokinetic characteristics of the compound of formula (I) in beagles.

[0222] The clear solution of the compound of formula (I) was injected into two 10-12 kg beagles via the cephalic vein or saphenous vein, and the clear solution of the compound of formula (I) was gavage administered to two 10-12 kg beagles (fasted overnight). Approximately 500 μL of blood was collected from the peripheral vein of the animals at 0.0333, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration, and transferred to a commercial centrifuge tube containing 0.85-1.15 mg of K2 EDTA*2H2O anticoagulant, and centrifuged at 4°C and 3000g for 10 minutes to obtain plasma. The blood drug concentration was determined by LC-MS / MS, and WinNonlin TM Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software was used to calculate relevant pharmacokinetic parameters using the non-compartmental linear logarithmic trapezoidal method.

[0223] Table 5 Pharmacokinetic parameters of the compounds in beagle dogs

[0224]

[0225] The active ingredient in the pharmaceutical composition of the present application has a high in vivo plasma clearance rate, a low oral plasma systemic exposure and a low oral bioavailability.

[0226] Experimental Example 6: Inhibition of the activity of human liver microsomal cytochrome P450 isozymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4)

[0227] A total of five specific probe substrates of five CYP isozymes, namely, phenacetin (CYP1A2), diclofenac (CYP2C9), (S)-mephenytoin (CYP2C19), dextromethorphan (CYP2D6) and midazolam (CYP3A4), were incubated with human liver microsomes and the compound of formula (I), and the reaction was initiated by adding reduced nicotinamide adenine dinucleotide phosphate (NADPH). After the reaction, the samples were processed and the concentrations of five metabolites produced by specific substrates, including acetaminophen, 4'-hydroxydiclofenac, 4'-hydroxymephenytoin, dextrorphan, and 1'-hydroxymidazolam, were quantitatively detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to calculate the corresponding half inhibitory concentration (IC 50 ).

[0228] Table 6 Inhibition parameters of the compounds of formula (I) on five CYP enzymes

[0229]

[0230] The active ingredients in the pharmaceutical composition of the present application have low inhibitory effects on five isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomal cytochrome P450.

[0231] Experimental Example 7: Study on drug efficacy in smoke-induced acute lung injury model in rats

[0232] Experimental animals

[0233] Male Sprague-Dawley rats (provided by Shanghai Slake Laboratory Animal Co., Ltd.), SPF grade, weighing about 200 g.

[0234] Experimental Procedure

[0235] 1. After arriving at the facility, the animals were fed for one week and randomly divided into 6 groups according to body weight;

[0236] 2. On the first to third days of the experiment, the corresponding compounds of each group were atomized for 30 minutes, and then the animals in the model group and each compound treatment group were exposed to cigarette smoke for 1 hour. After an interval of 4 hours, they were exposed to cigarette smoke again for 1 hour. The animals in the control group were exposed to cigarette smoke twice a day for 3 consecutive days.

[0237] 3. On the fourth day of the experiment, the corresponding compounds of each group were nebulized for 30 minutes. The animals in the model group and each compound treatment group were exposed to 150 μg / mL LPS by nebulization for 15 minutes, and 3 hours later (from the start of nebulization), they were exposed to smoke for 1 hour. The lung function (Penh and F) of the animals was tested again. After the animals were euthanized with CO2, the alveolar lavage fluid was collected for cell counting.

[0238] 4. Drug administration

[0239] Administration: Whole body exposure: The test compound and the reference compound were aerosolized using a nebulizer at the maximum aerosol rate (approximately 12 mL) for 30 minutes.

[0240] Dosing frequency: Nebulized drug or solvent 30 minutes before smoking every morning, and before LPS nebulization inhalation on the 4th day.

[0241] 5. Pharmacodynamic endpoint measurement

[0242] (1) Total white blood cell count in BALF (alveolar lavage fluid);

[0243] (2) Lung function test under methacholine (Mch) induction (airway resistance index Penh);

[0244] Table 7 Experimental groups

[0245] Group Number of animals Compound concentration of atomized solution Time of administration Model Group 10 - 30 minutes before the first smoking of the day Low dose group of compound of formula (I) 10 0.05mg / mL 30 minutes before the first smoking of the day High dose group of compound of formula (I) 10 0.15mg / mL 30 minutes before the first smoking of the day

[0246] The experimental results are shown in Figure 5 and Figure 6 .

[0247] Depend on Figure 5 and Figure 6 It can be seen that the active ingredients in the pharmaceutical composition of the present application can effectively reduce the number of white blood cells and the airway resistance index Penh in the alveolar lavage fluid.

[0248] Experimental Example 8: In vitro detection of the inhibitory activity of compounds on TNF-α in human peripheral blood mononuclear cells

[0249] Experimental purpose: To express the anti-inflammatory activity of the test compound at the cellular level based on the level of TNF-α in human peripheral blood mononuclear cells (hPBMC).

[0250] Steps:

[0251] 1. Collect whole blood from normal subjects and use EDTA anticoagulation tubes for anticoagulation;

[0252] 2. Ficoll density gradient centrifugation was used to separate PBMCs, count the cells, and adjust the cell concentration to 2x10 6 / mL;

[0253] 3. U-bottom 96-well plate, add 2×10 5 cells, LPS 1ng / mL, the compound of formula (I) was prepared into DMSO solutions with concentrations of 100μM, 10μM, 1μM, 100nM, 10nM, 1nM, 100pM, and 10pM, respectively, with 200μL system in each well;

[0254] 4. Cultivate for 24 hours and collect the supernatant;

[0255] 5.ELISA was used to detect the level of TNF-α in the supernatant, and Graphpad Prism software was used to fit the inhibition curve and calculate the IC 50 .

[0256] The experimental results are shown in Table 8:

[0257] Table 8 In vitro test results of compounds

[0258]

[0259] Therefore, the active ingredients in the pharmaceutical composition of the present application can exhibit effective anti-inflammatory activity and have a significant inhibitory effect on TNF-α in human peripheral blood mononuclear cells (hPBMC).

[0260] Experimental Example 9: Stability Test

[0261] The product obtained in Example 28 was placed under accelerated conditions (40°C±2°C / RH 25%±5%) and long-term conditions (30°C±2°C / RH 65%±5%) for 6 months. The results are shown in Table 10.

[0262] Table 9

[0263]

[0264] It can be seen that the pharmaceutical composition of the present application exhibits good stability under accelerated conditions and long-term conditions, with no significant increase in impurities and no increase in particle size.

Claims

1. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, and a buffer, 2. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, and an osmotic pressure regulator, 3. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, and a diluent, 4. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a surfactant, wherein the compound of formula (I) is a crystalline form of the compound of formula (I), 5. The pharmaceutical composition according to any one of claims 1 to 4, further comprising a metal chelator; Optionally, the metal chelator is one or more selected from edetic acid, edetate disodium or edetate calcium sodium; Optionally, it further comprises a buffer; Optionally, the buffer is one or more selected from sulfuric acid, hydrochloric acid, sodium hydroxide, citric acid, sodium citrate, lactic acid, sodium lactate, acetic acid, sodium acetate, trisodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, tartaric acid, sodium tartrate, glycine, boric acid, and phthalic acid; Alternatively, the buffer is selected from citric acid, citrate, tartaric acid, tartrate, phosphoric acid or phosphate; Alternatively, the buffer is selected from citrate, tartrate or phosphate; Alternatively, the concentration of the buffer is from about 0.01 to about 50 mg / mL; Alternatively, the buffer is used to control the pH value of the pharmaceutical composition to be between about 3.0 and about 8.5; Optionally, it further comprises an osmotic pressure regulator; Optionally, the osmotic pressure regulator is one or more selected from sodium chloride, potassium chloride, glucose, mannitol or xylitol; Optionally, it further comprises a diluent; Optionally, the diluent is one or more selected from water, ethanol and glycerol; Optionally, wherein the surfactant is a nonionic surfactant; Alternatively, the surfactant is selected from one or more of ethylene glycol polyoxyethylene ether, polypropylene glycol alkyl ether, alkyl polyglucosides, octylphenol polyoxyethylene ether, alkylphenol polyoxyethylene ether, glycerol alkyl ester, polyoxyethylene sorbitan fatty acid ester, sorbitan alkyl ester, sorbitan fatty acid ester, cocamide MEA, cocamide DEA, dodecyl dimethyl amine oxide, block copolymers of polyethylene glycol and polypropylene glycol, and polyethoxylated tallow amine; Alternatively, the surfactant is selected from one or more of Tweens and Spans; Alternatively, the surfactant is selected from one or more of Tween 20, Tween 80 and Span 20; Alternatively, the concentration of the surfactant is from about 0.01 to about 8 mg / mL; Optionally, the mass ratio between the compound of formula (I) or its pharmaceutically acceptable salt and the surfactant is about 1:200 to 100:1, preferably about 1:150 to 50:1, more preferably about 1:50 to 25:1, and even more preferably about 1:1 to 15:1, the mass of the compound of formula (I) or its pharmaceutically acceptable salt is calculated based on the compound of formula (I).

6. The pharmaceutical composition according to any one of claims 1 to 5, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, and at least one of a buffer, an osmotic pressure regulator, a metal chelator, and a diluent; Optionally, it comprises a compound of formula (I), a surfactant, a buffer, an osmotic pressure regulator, a metal chelator and a diluent, wherein the concentration of the compound of formula (I) is 0.002 to 50 mg / mL, the concentration of the surfactant is 0.02 to 3 mg / mL, the concentration of the buffer is 0.1 to about 25 mg / mL, the concentration of the osmotic pressure regulator is 5 mg / mL to 9 mg / mL, and the concentration of the metal chelator is 0.01 to about 5 mg / mL.

7. A pharmaceutical composition as described in any one of claims 1 to 6, comprising a compound of formula (I), Tween 80, sodium dihydrogen phosphate or its monohydrate, sodium hydrogen phosphate, sodium chloride, disodium edetate and water; optionally, the concentration of the compound of formula (I) is 0.002-50 mg / mL, the concentration of Tween 80 is 0.02 to 3 mg / mL, the concentration of sodium dihydrogen phosphate and disodium hydrogen phosphate is 0.1 to 25 mg / mL, the concentration of sodium chloride is 5 mg / mL to 9 mg / mL, and the concentration of disodium edetate is 0.01 to about 5 mg / mL.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the compound of formula (I) is a crystal of the compound of formula (I), and the crystal has an X-ray powder diffraction pattern using Cu Kα radiation, comprising 5, 6, 7, 8, 9, 10 or 11 diffraction peaks selected from the following 2θ angles: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 20.83±0.2°, 24.73±0.2° and 26.13±0.2°; or, using Cu The X-ray powder diffraction pattern of Kα radiation has diffraction peaks at the following 2θ angles: 5.81±0.2°, 13.96±0.2°, 15.01±0.2°, 17.95±0.2° and 24.73±0.2°; or, has diffraction peaks at the following 2θ angles: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 20.83±0.2°, 24.73±0.2° and 26.13±0.2°.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the particle size of the compound of formula (I) or a pharmaceutically acceptable salt thereof is X 50 ≤10μm; Alternatively, the particle size of the compound of formula (I) or its pharmaceutically acceptable salt is X 50 ≤5μm and X 90 ≤10μm.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the pharmaceutical composition is in the form of a suspension.

11. Use of the pharmaceutical composition according to any one of claims 1 to 10 in the preparation of a medicament for preventing or treating a disease associated with PDE3 and / or PDE4; optionally, the disease associated with PDE3 and / or PDE4 is selected from asthma or chronic obstructive pulmonary disease.

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